phosphorus-radioisotopes and 3-4-epoxy-1-butene

The reaction of 3,4-epoxy-1-butene (BMO) with deoxyguanosine-3'-monophosphate (3'-dGMP) resulted in the formation of two pairs of diastereomeric 7-alkyl-3'-dGMP derivatives corresponding to two isomers C¿-1 and C¿-2. The T4 polynucleotide kinase-mediated phosphorylation with [gamma-32P]-ATP showed preferential labelling of diastereo- mers of the C¿-1 isomer. The diastereomers 1 and 2 of the C¿-1 isomer had labelling efficiencies of 42%. However, the labelling efficiencies of diastereomers 3 and 4 of the C¿-2 isomer were 11 and 10%, respectively. The 32P-postlabelling of BMO-modified DNA yielded four isomers in the ratio of 4:4:1:1 with overall recoveries being 14%. The two isomers had a half-life of 270 min (C¿-1 isomer) and 300 min (C¿-2 isomer) which is in accordance with the stability predicted by other similar adduct experiments. The molecular modelling experiments showed more pronounced restricted rotation of butadiene residue in C¿-2 isomers due to steric interaction between butadiene residue at N-7 and O(6) atom of guanine than in C¿-1 isomer. The butadiene residue also leads to steric overcrowding at 3'-phosphate in C¿-2 isomer which probably restricts the access to the active site of T4 polynucleotide kinase.

Topics: Animals; Binding Sites; Deoxyguanine Nucleotides; DNA; DNA Adducts; Epoxy Compounds; Half-Life; Isotope Labeling; Male; Models, Molecular; Mutagens; Phosphorus Radioisotopes; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase; Salmon; Stereoisomerism

In this paper we report DNA binding of butadiene monoepoxide, a first metabolite of 1,3-butadiene catalyzed by monooxygenases. We prepared alkylated purines as marker compounds for 32-P-postlabeling and electrochemical analysis and developed methods to measure the corresponding products. The traditional postlabeling assay was modified by incorporating a solid phase extraction column and high-performance liquid chromatography (HPLC) enrichment steps to the assay prior to labeling. The final analysis of adducted N6 adenines is based on two dimensional thin-layer chromatography (TLC) and an on-line HPLC/radioactivity analysis. The qualitative and quantitative results are based on positively identified marker compounds. Alkylated N7 guanines were released from DNA by neutral thermal hydrolysis, prepurified by HPLC, and analyzed by HPLC with a sensitive electrochemical detection procedure. By using these methods, we found alkylation of calf thymus DNA exposed to butadiene monoepoxide in vitro at adenine N6 and guanine N7 sites. Analysis of lung DNA samples from mice and rats exposed to butadiene through inhalation showed that adenine N6 adducts were formed in vivo in a dose responsive manner.

Topics: Adenine; Administration, Inhalation; Alkylation; Animals; Butadienes; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA Adducts; Epoxy Compounds; Guanine; In Vitro Techniques; Mice; Phosphorus Radioisotopes; Rats

Butadiene monoepoxide, an active metabolite of 1,3-butadiene, was reacted with deoxyadenosine, deoxyadenosine 3'-monophosphate and DNA. The nucleoside reaction products were isolated and using various spectroscopic techniques were determined to be the N6-substituted deoxyadenosine adducts. Deoxyadenosine 3'-monophosphate products were identified by treating the modified nucleotide products with alkaline phosphatase, resulting in nucleoside adducts with HPLC retention times similar to those of the deoxyadenosine adducts. Monophosphate products were also identified through MS/MS techniques by comparing the daughter ions derived from the base moieties of N6-alkylated nucleosides and nucleotides. The reaction mechanism in aqueous solution was studied using optically active butadiene monoepoxides. Using the alkylated monophosphate standards and an HPLC/32P postlabeling assay the N6-alkylated adenine adducts were detected in calf thymus DNA exposed to butadiene monoepoxide.

Topics: Adenine; Animals; Cattle; Chromatography, High Pressure Liquid; DNA; Epoxy Compounds; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phosphorus Radioisotopes

We have developed an HPLC-32P-postlabelling procedure to detect DNA adducts formed by epoxybutene and diepoxybutane. The method exploits the interaction of the two epoxides with deoxynucleotides and polydeoxynucleotides to optimize the HPLC enrichment of adducted nucleotides before 32P-postlabelling. Using this approach, a number of guanine adducts were identified after the exposure of dGMP, poly(dG-dC) or calf thymus DNA to epoxybutene and diepoxybutane, and a major adenine adduct was identified in poly(dA-dT) and calf thymus DNA exposed to diepoxybutane.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; DNA; DNA Damage; Environmental Monitoring; Epoxy Compounds; Evaluation Studies as Topic; Humans; In Vitro Techniques; Mutagens; Phosphorus Radioisotopes